From: Peter Schuck <pschuck@helix.nih.gov>
  To  : Houphouet Hy Yarabe <hyarabe@unix1.sncc.lsu.edu>
  Date: Fri, 30 Apr 1999 09:55:32 -0700

Re: Molecular weight (Mw)

Dear Yarabe, 

I completely agree with your assesment that the diffusion coefficients from
sed velocity frequently lack accuracy.  This is an ill-conditioned problem.
 I have calculated the contours of the error surfaces for s and D (or M)
from data at different speeds and solution columns (Biophys. Journal (1998)
75:1503-1512), which documents this (Figure 4).  

The best test for consistency and accuracy is whole boundary fitting of
your sedimentation velocity data with a Lamm equation fitter (such as
sedfit, a Windows95 software for fast Lamm equation fitting), where you can
fit the sedvelocity data by constraining M to the value that you know from
your equilibrium measurements.  (The software allows you to transform the
parameter space from s,D into s,M, and allows you to constrain M).  This
would give you residuals for your fit to the sedvelocity data, and if the
residuals are acceptable, this demonstrates that your sedimentation
velocity analysis is in agreement with the equilibrium data, within the
given noise of data acquisition.  That's all there is to it, in my mind. 

If you get D from boundary spreading alone, as suggested by your reviewer,
you might get a wrong value of D by virtue of the analysis being an
ill-conditioned problem.  This would generate an apparent inconsistency
which could completely lack any reality.  

Please don't hesitate to let me know if you have any further questions. 

Best wishes, 
Peter


At 11:21 PM 4/29/99 -0500, you wrote:
>I reported Mw by sedimentation equilibrium and S values determined by
>sedimentation velocity as part of a paper I've submitted to Analytical
>Chemistry. The paper got some good reviews.  However, one of the reviewers
>wants me to consider adding values of diffusion coefficient (D) which can
>be obtained from the width of sedimenting boundaries measured in the
>sedimentation velocity.  With D values, he adds, Mw results can be
>calculated using the Svedberg equation and compared with Mw obtained via
>sedimentation equilibrium.  This comparison, he concludes, would provide
>an excellent test of the accuracy of reported s and Mw (sedimentation
>equilibrium) values.
>
>I aggree with him on the principle.  However, in my mind, the diffusion
>coefficients calculated from sedimentation velocity lack accuracy. Could
>somebody give me some input to address this reviewer.
>
>"Struggle to know more ....."
>
>Yarabe
>
>Louisiana State University
>Baton Rouge, LA 70803
>Tel: 225-388-3919
>Fax:  225-388-3971
>   

***********************************************************
Peter Schuck, PhD
Molecular Interactions Resource
Bioengineering and Physical Science Program, ORS
National Institutes of Health
Bldg. 13 Rm. 3N17
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Bethesda, MD 20892 - 5766
Tel: (301) 435-1950
Fax: (301) 496-6608
email: Peter_Schuck@nih.gov
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